Aqueous Two Phase Systems (ATPS) have been employed in several areas of biotechnology, such as in the purification and isolation of biomolecules, cells, and organelles, as well as in bioconversion processes and diagnostic applications. These applications rely on the fact that most biological molecules tend to distribute unevenly between the two liquid phases (where the ratio of the solute concentrations in the upper to lower phases in such phase systems is termed the partition coefficient, ("K").
In cases where the partition coefficient of a particular substrate does not favour efficient separations (i.e., where K is close to unity), the physical parameters of the system may be suitably adjusted to enhance the partitioning by varying parameters such as pH, temperature, polymer nature and concentration, salt, and the like. Of particular interest are the many advantages this technique offers in the large-scale purification of enzymes and proteins in comparison to conventional liquid-solid based separation techniques, such as chromatography. The theoretical and practical aspects of this technique have been described, for example, in Walter, H., Brooks, D. E., and Fisher, D., Partitioning in Aqueous Two-Phase Systems, Academic Press, New York, 1985; Andersson, E., Johansson, A.-C. and Hahn-Barberdal, B., Ann. N. Y. Acad. Sci., 415, 115-118, 1985, Aqueous Two-Phase Systems for Producing Alpha-Amylase Using Bacillus subtilis; Larson, M. and Mattiasson, B., Ann. N. Y. Acad. Sci., 415, 144-147, 1985, Continuous Conversion of Starch to Ethanol Using a Combination of an Aqueous Two-Phase System and an Ultrafiltration Unit, Mattiasson, B. and Larson, M., UK Pat. Appl. GB 2,168,617A, 1986). The prior art has employed aqueous solutions of incompatible polymer pairs in the ATPS. Another approach has utilized aqueous polymer/salt systems, e.g., polyethylene glycol/salt, for large scale purifications. However, the presence of high salt concentrations may have undesirable effects (such as denaturation) on many biological substrates, such as proteins and in affinity partitioning, and may therefore not provide a versatile and generally applicable method.
Various incompatible polymer pairs have been reported in the literature, including dextran/polyethylene glycol (PEG), PEG/polyvinyl pyrrolidone (PVP), polypropylene glycol (PPG)/polyvinyl alcohol (PVA), PVA/dextran, PVA/methyl cellulose, PVP/methyl cellulose, PPG/PVP, PPG/dextran, PEG/Ficoll, and other systems based on combinations of derivatives of dextran or starch and synthetic polymers. Much of the prior art has been concerned with the use of dextran/PEG phase systems in view of various practical limitations (e.g., high solution viscosities, long settling times, instability of the polymers, etc.) of many of the above cited polymer combinations. However, a major drawback from a commercial aspect is the high cost of fractionated dextran in these systems, which tends to preclude most large-scale applications.